Application of photoacoustic computed tomography in biomedical imaging: A literature review

Abstract Photoacoustic computed tomography (PACT) is a hybrid imaging modality that combines optical excitation and acoustic detection techniques. It obtains high‐resolution deep‐tissue images based on the deep penetration of light, the anisotropy of light absorption in objects, and the photoacoustic effect. Hence, PACT shows great potential in biomedical sample imaging. Recently, due to its advantages of high sensitivity to optical absorption and wide scalability of spatial resolution with the desired imaging depth, PACT has received increasing attention in preclinical and clinical practice. To date, there has been a proliferation of PACT systems designed for specific biomedical imaging applications, from small animals to human organs, from ex vivo to in vivo real‐time imaging, and from simple structural imaging to functional and molecular imaging with external contrast agents. Therefore, it is of great importance to summarize the previous applications of PACT systems in biomedical imaging and clinical practice. In this review, we searched for studies related to PACT imaging of biomedical tissues and samples over the past two decades; divided the studies into two categories, PACT imaging of preclinical animals and PACT imaging of human organs and body parts; and discussed the significance of the studies. Finally, we pointed out the future directions of PACT in biomedical applications. With the development of exogenous contrast agents and advances of imaging technique, in the future, PACT will enable biomedical imaging from organs to whole bodies, from superficial vasculature to internal organs, from anatomy to functions, and will play an increasingly important role in biomedical research and clinical practice.


| INTRODUCTION
Photoacoustic computed tomography (PACT) is a major manifestation of photoacoustic tomography (PAT). 1 PACT is based on the photoacoustic effect, where biological tissues absorb the incident light, and generates acoustic waves in ultrasound range, the acoustic waves are detected by traditional ultrasound transducers, and crosssectional and 3D images are finally reconstructed from the acoustic waves. 1 Recently, there has been increasing interest surrounding the use of PACT in biomedicine. PACT, as a hybrid imaging modality, achieves light absorption measurement and acoustic detection simultaneously. [2][3][4] While the resolution and imaging depth of optical imaging modalities such as confocal microscopy, two-photon microscopy, and optical-coherence tomography are limited by the diffraction barrier and optical diffusion limit, respectively, 1,5 PACT overcomes the diffraction and diffusion limits of these imaging modalities to enable imaging with deep penetration, high spatiotemporal resolution, and rich contrast. [1][2][3][4] Currently, the biomedical applications of PACT include both preclinical research and clinical applications. 1 Preclinical research focuses on small animals, such as brain imaging, whole-body vascular density imaging, and whole-body functional imaging of mice. 6,7 Jose et al. performed hybrid imaging of light absorption, speed of sound, and acoustic attenuation in 2010. 8 Xia et al. reported a novel small-animal whole-body imaging system called ring-shaped confocal PACT (RC-PACT) in 2012, which provides a series of cross-sectional images of the mouse brain, liver, kidney, and bladder. 7 Using the RC-PACT system, Chatni et al. demonstrated that both anatomy and glucose uptake can be imaged with a single modality. 9 Yao et al. demonstrated the feasibility of imaging mouse brain metabolism using PACT in 2012. 10 Meng et al. developed ultrasonic-array-based opticalresolution PACT in 2012. 11 Xia et al. introduced retrospective respiratory gating for whole-body PACT in 2014. 12 In 2016, Li et al. demonstrated that PACT can provide high-resolution label-free imaging of structures in the entire mouse brain. 13 Mitsuhashi et al. first proposed transcranial PACT brain imaging in 2017. 14 In the same year, Li et al. imaged in vivo whole-body dynamics of small animals by using single-impulse panoramic PACT and achieved high spatiotemporal resolution. 6 In 2018, Wang et al. found that ingestible baked barley can enhance the contrast of photoacoustic imaging. 15 Later, Li et al.
imaged both a 7.5-cm deep leaf target embedded in an optically scattering medium and the beating heart of a mouse overlaid with 3.7-cm thick chicken tissue via internal light illumination combined with external ultrasound detection to improve the penetration depth of PACT. 16 In 2019, Liang et al. investigated the impact of the murine skull on the noninvasive PACT, which paved the way for future PACT of mouse brains. 17 Lv et al. achieved structural and functional imaging of ischemic stroke via a bowl-shaped array PACT in 2020. 18 Later in the same year, Li et al. introduced an ergodic relay into the PACT system, which had the potential for functional imaging and biometric application in vivo. 19 In 2021, Huang et al. used multispectral optoacoustic tomography (MSOT) to visualize lipids in laboratory mice, and demonstrated that MSOT was an efficient imaging technique for lipid visualization in the preclinical model of fatty liver disease. 20 In 2022, Vagenknecht et al. demonstrated tau protein imaging in mice brain using tau-targeted pyridinyl-butadienyl-benzothiazole derivative PBB5 and MSOT. 21 Clinical research of PACT mainly focuses on the examination of human breast cancer and skin melanoma. 22, 23 Wang et al. demonstrated that compact lasers based on emerging diode technologies are well suited for preclinical and clinical PACT. 24 Jose et al. studied the applicability of PACT imaging as an intraoperative modality for examining the status of resected human sentinel lymph nodes in 2011. 23 Es et al. investigated the use of PACT to visualize the blood vessels in a healthy human finger, focusing on vascularity across both interphalangeal joints, in 2014. 25 Biswas et al. created a method for delineating surfaces of the finger's bone, which can be used to detect rheumatoid arthritis, in 2015. 26 Meng  MPAUCT, which combines multispectral PACT and ultrasound computed tomography for imaging of human finger joints. 28 Lin et al. achieved PACT imaging of human breast tissue by scanning the entire breast within a single breath-hold (SBH; 15 s) in 2018, which was the first time PACT had been used for human breast cancer detection.
They imaged the breasts of seven breast cancer patients and identified seven of eight tumors successfully. 22 Via a self-developed PACT system, Wray et al. obtained volumetric image of angiographic structures in human extremities including arms and legs in 2019. 29 Later that year, Yang et al. used a co-registered PACT and ultrasound system to image ex vivo human colorectal tissue samples, and showed the potential of PACT in the diagnosis of colorectal cancer. 30 In 2020, Yang et al. developed a concave transducer array-based PACT system, and realized hemodynamic monitoring of the foot vessels in diabetic patients via the PACT system. 31 Shan et al. demonstrated the feasibility of PACT in monitoring fetal vascular dynamics in the same year. 32 In 2021, Liapis et al. used an eigenspectral MSOT to evaluate chemotherapeutic effects on breast tumor hemodynamics. 33 Via massively parallel transducers-based PACT system, Na et al. achieved human brain functional imaging. 34 PACT has emerged as a novel imaging modality that provides high-resolution imaging of optical absorption in deep tissue. Over the past few years, various preclinical and clinical applications of the technique have been demonstrated, including functional brain imaging, small-animal whole-body imaging, breast cancer screening, and guidance of lymph node biopsy. 1 In this review, we searched the Web of Science database for articles published during the past two decades using keywords including PACT, biomedical, animal, and human. And the article reviews the applications of PACT in biomedical research and clinical practice with the following structure: Section 2 introduces PACT, contrast agents and reconstruction algorithms.  An optical parametric oscillator pumping laser is typically used as the light source in PACT systems because it can deliver nonionizing laser pulses to the object and lead to thermal expansion. A light path system is designed for diffracting or focusing a beam of light, and it may include a linear microlens array or a diversified objective lens depending on the imaging goal. The light beams are focused on the imaging platform where the target imaging object is placed. Then, the acoustic transducer measures conventional acoustic signals from the object. Finally, the image is reconstructed from the received PA signals via various algorithms in the post-processing platform. 8

| PACT systems applied in biomedical imaging
UP to now, several PACT systems with different configurations have been proposed and used in biomedical imaging. According to the perpendicular or parallel relationship between incident light and the signal receiving surface of the detector, PACT systems can be roughly classified into two types [6][7][8]11,22,35 (Figure 1). The transducer of the former (Type I) is usually rectangular or arc-shaped, volumetric images are obtained by rotating the transducers or rotating the imaging object 8,11,20 (Figure 1a). The transducer of the latter (Type II) is usually ring-shaped to obtain cross-sectional images. 6,7,22,35 Three-dimensional images are obtained by scanning the imaging object elevationally 6,7,22,35 (Figure 1b).
In terms of the number of acoustic transducers, Type I PACT usually contains dozens of elements in the transducer array, 8,11,20 Type II PACT contains several hundreds of elements in general. 6,7,22,35

| Endogenous and exogenous contrast agents
Since the amplitude of the PA signal is sensitive to optical absorption, variations of biological tissues in optical absorption introduce imaging contrast in PACT imaging. 44,45 In biomedical PACT imaging, materials that absorb optical signal can be endogenous or exogenous, and are known as endogenous or exogenous contrast agents. 45

| Endogenous contrast agents
Endogenous contrast agents are biomolecules inside the body; therefore, the main advantage of imaging them with PACT is the label-free capability with high optical absorption contrast. 45 Endogenous chromophores mainly include hemoglobin, melanin, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), lipids, and water. 45 Hemoglobin is the dominant absorber of light in the visible and near-infrared (NIR) part of the optical spectrum and is commonly used for PACT imaging. 46 Imaging hemoglobin with PACT can not only visualize vascular anatomy, 4 but also provide functional information associated with blood oxygen saturation (sO 2 ). [47][48][49] Melanin is another important endogenous absorber, which is sensitive to visible and NIR light around 750 nm. 4,22,23 Since melanin is produced by melanocytes, it can be used to reveal melanomas and metastatic melanoma cells. 50 20 Lipids have strong optical absorptions around NIR wavelengths, with the absorption peak occurring at 930 nm. 45 Imaging lipids with PACT has enabled the visualization of hepatic steatosis in a preclinical mice model, 20 breast tumors, 52 and arterial walls. 53 Water absorbs strongly at NIR wavelengths longer than 900 nm, with a strong peak at 980 nm. 45  To overcome this issue, target-specific exogenous contrast agents for PACT have been developed by researchers. 54 The most common type of exogenous contrast agents is nanoparticle. 55 To date, various nanoparticles have been designed for PACT imaging. 55,56 Metallic nanoparticles, especially gold nanoparticles including spheres, rods, shells, and cages are commonly used contrast enhancers in PACT for imaging specific biological processes, such as inflammatory response, 57,58 tumor, 54 macrophages, 59 and melanoma. 60 In addition to gold, other metallic nanoparticles, such as silver, platinum, copper, and palladium have been used to generate contrast in PACT imaging. 45,55,61 Carbon nanotubes are another class of nanoparticles that have been used as contrast enhancers to improve contrast and sensitivity of tumor and angiogenesis imaging in mice. 62,63 Although nanoparticles are commonly used in PACT, concerns remain as they may lead to potential cytotoxicity and inflammatory response. 45 Organic contrast agents overcome the disadvantages of the metallic nanoparticles and carbon nanotubes, as they are nontoxic and biocompatible. 54,64,65 Some organic dyes absorb NIR light and can lead to enhanced contrast in PACT imaging. 54 For instance, via indocyanine green-based contrast enhancement, target-specific PACT tumor imaging has been realized. 66,67 Methylene blue has been assisted to visualize lymph nodes in humans and rats. 64,68,69 Other dye-based exogenous contrast agents such as Evans blue, phthalocyanine have been used in PACT imaging. 44 Organic contrast agents such as semiconducting polymer, semiconducting small molecules have also been used to fabricate NIR-I and NIR-II PACT contrast agents. 55,64,65,70,71 In addition, via some organic contrast agents, protein imaging can be achieved. For instance, tau-targeted pyridinyl-butadienyl-benzothiazole derivative PBB5 can aid tau-protein imaging in the mice brain. 21 There are also protein-based phytochromes, such as RpBphP1, RpBphP3, and DrBphP phytochromes, which are binded with some heme-derived biliverdin chromophore and can aid the detection of protein-protein interactions in tumors and livers of mouse models via RS-PACT and SIP-PACT configurations. 38,72

| PACT imaging reconstruction algorithm
In a PACT system, acoustic transducers are used to collect the PA signals from biological tissues, and then reconstruction algorithms are applied to reconstruct light absorption images. 73 Currently, reconstruction methods for PACT can be divided into two main categories: conventional reconstruction methods and learning-based reconstruction methods. 74 Conventional reconstruction methods include direct reconstruction (back-projection algorithms and filtered back-projection algorithms) 75 under the condition of sparse sampling. 73 In addition, direction reconstruction methods usually ignored acoustic heterogeneity and used a constant speed of sound for image reconstruction. 80 Model-based iterative reconstruction aims to reconstruct the PACT images by solving the optimization problem iteratively. 74 This reconstruction technique can address the inhomogeneous acoustic properties of the imaging object, 77 such as dual-speed reconstruction, 6 joint reconstruction, 81 and ultrasound computed tomography enhanced reconstruction. 82 Modelbased iterative reconstruction methods usually assume multiple speed of sound during image reconstruction, which efficiently address the acoustic heterogeneity. 80 However, model-based iterative reconstruction is computationally intensive. 73,74,83 Learning-based reconstruction methods for PACT are based on machine learning algorithms such as directory learning and deep learning algorithms. 73,74,83 Learning-based reconstruction methods can learn endto-end transformation from both direction reconstructions and model-based iterative reconstructions, and can further reduce the artifacts and noise generated from the conventional reconstruction methods. 74 To date, several deep learning algorithms including U-Net and its variants, 77,83 Y-Net and its variants, 74,84 have been developed for PACT image reconstruction with enhanced image quality and spatial resolution. 80 Recently, generative adversarial network (GAN) and its variants including Wasserstein GAN, knowledge infusion GAN, 74,83,85 have been used to approximate the real PA data, and have achieved superior performances in terms of signal-to-noise ratio and structural similarity. 83

| THE APPLICATION OF PACT IN ANIMAL IMAGING
With the wide use of model organisms in biomedical and preclinical studies, in vivo tissue imaging and whole-body imaging are becoming  These studies have proved that PACT can overcome the effect of ultrasound signal attenuation through relatively thick skulls and that animal cerebral cortex imaging is feasible via PACT. By implementing external and internal illuminations simultaneously, PACT can potentially achieve whole-brain imaging, thereby providing a new way to study the mechanism of cerebral blood supply. Furthermore, the application of PACT as a rapid imaging modality on brain metabolism has been realized. 10 can provide whole-body imaging, as well as in vivo imaging of the brain, chest, breast, heart, liver, stomach, intestine, and kidney of the rodent (as shown in Figure 2). 10,20,[92][93][94][95][96][97][98][99][100][101][102] Using NIR light, PACT can provide whole-body imaging of animals with acoustically defined spatial resolution. 6,7,12 Via endogenous hemoglobin contrast, anatomical and vascular structures can be imaged, and by using the wide choice of exogenous optical contrasts, functional, and molecular imaging can be enabled. 45 Table 2 summarizes the applications of PACT on animal imaging studies. Since a majority of biomedical and preclinical studies have focused on the brain and vascular system, we will provide detailed reviews on PACT imaging of the brain and cardiovascular system in animals. In addition, as there are increasing attentions on adipose tissue, we also review the application of PACT on adipose tissue imaging.

| PACT imaging of animal brain
The main application of PACT in model animals is brain imaging. Since In addition to its rich applications in displaying brain structure, vascular networks, and hemodynamics, PACT also excels in functional and metabolic brain imaging (the middle two subfigures of Figure 2b). 93,103 Back in 2013, using 2-NBDG as the exogenous contrast and hemoglobin as the endogenous contrast, Yao et al. demonstrated that PACT is capable of imaging the metabolic response of a mouse brain to forepaw stimulations, as shown in the right subfigures of Figure 2b. 9 Tang et al. also demonstrated that PACT could be used for monitoring the cerebral responses in behaving rats to sensory stimulus. 93 Later, researchers confirmed noninvasive, unlabeled, and functional PACT in rat brain for accurately mapping brain injury and cerebral hemodynamics. 13 Whole-body PACT can obtain anatomical structures and detailed images of the vascular structures within organs. Moreover, whole-body PACT images with improved image quality after respiratory gating can be used to monitor the pathological changes of internal organs, thus providing a promising approach to whole-body functional and metabolic imaging. system can image the beating hearts of mice under multiple overlays and makes it possible to image internal organs that are deeply seated in the human body and relatively close to body cavities. 16

| PACT imaging of animal adipose tissue
Recently, the study of adipose tissue has attracted increasing attention, due to the association between adipose tissue with a variety of diseases, including obesity, diabetes, and cachexia. 20 [116][117][118] With noninvasive, label-free, and radiation-free features, PACT is friendly to patients, medical staff, and clinical application.  29 The MAP of the PA signal along the Z-axis is presented on the left, and a color-coded depth image of the hand is presented on the right. (k) MSOT images with increased signals for oxyhemoglobin and sO 2 in the intestinal wall of a patient with Cohn's disease. 111 lipomas at different areas of six subjects used a clinical MSOT system. 119 Clinical MSOT can also image BAT based on hemoglobin gradients, and can detect BAT activation after cold exposure by the increase of PA signal, as shown in Figure 3c. 104 Owing to the easy accessibility of human skin, PACT imaging of skin-related diseases, especially skin cancer has gained intense attention. As displayed in Since the human skin is easily accessible, PACT has been widely used in many preclinical and clinical dermatologic scans. Due to the unprecedented sensitivity of PA signals to hemoglobin and melanin, PACT has been commonly used for imaging the subcutaneous vascular system and melanoma. 47 PACT can provide high-resolution sO 2 measurements using hemoglobin as an endogenous contrast agent, which is significant because sO 2 is an important biomarker for skin cancer healing, treatment monitoring, and wound healing monitoring. 48

| Tumor imaging
PACT can provide morphological and functional information on early stage cancer using endogenous or exogenous contrast agents. 129 Angiogenesis plays an essential role in the development, invasion, and metastasis of cancer, which results in a locally abnormal vascular morphology and increased density. Structural and/or functional vascular changes and associated deficits in oxygenation are crucial indicators of early stage cancer and potential targets of relevant therapy. 130 PACT, as a powerful tool for label-free angiography, can achieve quantitative analysis of the spatial distribution, morphologic changes, and density of vessels, making it an effective approach for tumor imaging. 130 In addition, functional features such as blood flow velocity and blood sO 2 can also be obtained with PACT. 129 To date, in terms of animal models, deep tumor imaging, monitoring the effect of certain therapies have been realized, 65,131 and measurements of tumor microenvironments, including pH change, enzyme T A B L E 3 Summary of tumor imaging studies based on PACT

Regions Research details
Breast cancer Increases in vascular density and changes in blood sO 2 are key markers for breast malignancies. 129 Enabled by rapidly toggling laser wavelengths in the NIR spectral range, PACT is capable of whole breast functional imaging, yielding clear images of breast vessels and tumors simultaneously. 132 In addition to vascular density imaging, detecting relative changes of tissue components caused by tumors is another advantage of PACT for breast cancer screening. 129 By analyzing changes in the ratios of different forms of hemoglobin and the relative blood oxygenation, the spatial heterogeneity of a breast tumor can be quantified. 133 A recent study achieved volumetric imaging of human breast cancer in 15 s using the SBH-PACT system, with the real-time acquisition of 2D slices of breast anatomy, and demonstrated PA angiography with no breathing-induced motion artifacts. 22 PACT is able to provide depth-encoded PA vessel images of cancerous breast tissue showing the detailed vascular structure, and it is also able to present images of quantitative hemoglobin concentration and sO 2 in healthy and afflicted breasts to accurately visualize tumor regions. 22,134 Skin cancer Due to its inherent advantages, PACT holds great promise for skin cancer detection and quantification. 135 PACT uses endogenous tissue chromophores (e.g., melanin or hemoglobin) to provide a unique combination of optical absorption contrast and ultrasonic spatial resolution for attaining high-sensitivity images at centimeter-level depths. Therefore, in vivo images of deeper structures can be provided, enabling determination of tumor thickness and visualization of blood flow in skin vasculature. 136 In addition to being used for deep-tissue imaging and determining the thickness and structure of melanoma, PACT can be used to visualize lymph node metastases and detect circulating melanoma cells, allowing for the clinical evaluation of primary and metastatic malignant melanoma, noninvasive visualization of tumor boundaries, and assistance with the assessment of metastatic status, which can facilitate more effective treatment and better clearance of tumor cells and reduce the need for additional biopsies. 50 Prostate cancer With structural, functional, and molecular imaging characteristics, PACT is expected to be an effective imaging modality for the early diagnosis and treatment evaluation of prostate cancer. 72 In an ex vivo study, PACT was verified to differentiate malignant prostate tissue, benign prostatic hyperplasia, and normal human prostate tissue from prostate tissue sections by indicating the presence of deoxyhemoglobin, oxyhemoglobin, lipids, and water. 137 In vivo studies have demonstrated the feasibility of PACT for recognizing the angiogenesis of prostate cancer based on how the intensity of PA signals corresponds to vascular parameters (e.g., vessel length and vascular density). Therefore, PACT can be potentially applied for imaging prostate cancer angiogenesis by comparing the microvascularity between normal prostate tissue and cancerous prostate tissue. 138,139 Thyroid cancer Multispectral PACT has been performed on surgically excised thyroid tissue, and chromophore images representing the optical absorption of deoxyhemoglobin, oxyhemoglobin, lipids, and water have been reconstructed. Chromophore analysis on PA images can aid in differentiation of malignant from nonmalignant thyroid tissue with a sensitivity of up to 70%. [140][141][142] Ovarian cancer Oxyhemoglobin saturation, an indicator of tumor metabolism and therapeutic response, is also an important diagnostic measure of PACT. In cystic ovarian tissue, the PACT penetration depth may reach 6-7 cm, which enables imaging of more than 95% of the ovaries. 143 PACT can show the distribution of deoxyhemoglobin and oxyhemoglobin, contributing to the identification of both high-grade epithelial ovarian cancer and low-grade ovarian tumor. 144 Cervical cancer PACT is capable of intact scan both around the external orifice and in the cervical canal. In one previous study, depth MAP images obtained through in vitro experiments were analyzed to evaluate the extent of angiogenesis for different clinical stages of cervical cancer. The results revealed that the mean optical absorption of the cervical lesions was closely related to the severity of cervical cancer. 145 With the ability to distinguish cervical lesions at the depth of 5 mm, PACT may have great utility in the clinical diagnosis of cervical cancer. 145 Colorectal cancer PACT can be used to differentiate normal from tumor tissues in the colorectum through quantitative measurement of hemoglobin concentration and the spectral features. A pilot study reported PACT imaging of colorectal cancer samples ex vivo, and achieved area under the curve values of more than 0.95 in distinguishing normal from tumor tissues. 30 Abbreviations: NIR, near-infrared; PACT, photoacoustic computed tomography; SBH, single breath-hold.
activity, and reactive oxygen species levels have been achieved. 131 As for tumors in human body, there have been several PACT-based tumor imaging studies involving breast, melanoma, prostate, thyroid, ovarian, and cervical cancers, and the studies are summarized in Table 3. Since PACT imaging of skin cancer and breast cancer received much research attention, we will give a comprehensive review of these aspects in the following sections.

| PACT imaging of skin cancer
Skin and subcutaneous tissue are easy to assess for PACT. PACT imaging of skin and subcutaneous tissues reveals vascular structure, oxygenation, and flow. These parameters can provide information about the tumor microenvironment, such as angiogenesis and hypoxia, which are of clinical importance to the detection and assessment of skin cancer. 129 Among various skin cancers, melanoma is the most aggressive skin cancer. 91,129 Melanoma initially grows within the epidermis, which is followed by a vertical growth phase with deeper extension. 146 Therefore, noninvasive and quantitative depth assessment for melanoma is of clinical importance. 129 In addition, early detection of malignant melanoma and subsequent precise surgical extirpation can reduce the mortality of this fatal cancer.
PACT has excellent capabilities for melanoma detection and

| PACT imaging of breast cancer
Breast cancer is one of the most commonly diagnosed cancer in women. 127 Compared with other body parts, the breast has lower vascular density. The dense breast tissue has little effect on PA signals. 134 In addition, angiogenesis and changes in blood sO 2 and hemoglobin concentration are hallmarks of breast malignancy. 130 In 1994, the idea of using photoacoustic for breast imaging was proposed, and the first experiment was implemented in 2001. 134 Since then, many PACT configurations have been reported. 134 In summary, most of the existing PACT configurations used NIR-I or NIR-II lasers to identify features including sO 2 (Figure 5a), hemoglobin concentration (Figure 5b), 149,150 and to visualize vessel density, vasculature in the breast for tumor detection (Figure 5c). 132,151,152 In addition to morphological imaging, detection of relative changes in tissue composition due to tumor is another advantage of PACT in breast cancer screening. 129  Endogenous contrast agents are mainly used for structural imaging of tissues, particularly anatomical and vascular imaging of tissues. 7,12 Advanced functional imaging and molecular imaging require the use of exogenous contrast agents. 116 Exogenous agents can be prepared as required, with an increase of two to three orders of magnitude in molar absorption coefficients in specific wavelengths compared to endogenous contrast agents, which is of benefit to both dynamic imaging and kinetic imaging. 103 For example, IR-780 can be used to construct super-high-resolution cerebral vascular network structures for hemodynamic studies 103 ; 2-NBDG can be used for quantitative imaging of brain metabolism 10 ; hyaluronate-silica nanoparticle can be used as biocompatible liver-targeted PA contrast agents 153 ; indocyanine green can mark lung tumor cells 154 ; and gold nanoparticles can be used in the assistant diagnosis of early kidney damage and contrast imaging of cancer cells. 155 With stronger interdisciplinary collaborations between PACT and materials science researchers, more and more exogenous contrast agents for PACT will be developed in the future for target-specific functional and molecular imaging.
Since the advent of PACT, many photoacoustic imaging techniques have been developed to improve resolution and penetration depth, such as multispectral technique, multi-angle illumination technique, and so forth. 28,37 Internal illumination is a promising technique for PACT imaging of internal organs and tissues in preclinical and clinical practice. 156 Existing studies have demonstrated the use of internal illumination to image deep structures of the mouse brain and the beating heart of mice under 37 mm thick chicken breast tissue, 16 and they have shown the ability of internal illumination to realize PACT imaging to a depth of 10 cm through in vitro experiments and in vivo experiments involving renal vascular imaging in live pigs. 42 The combination of simultaneous external and internal illumination is expected to enable PACT imaging of the whole human brain, as well as the lungs, lymph nodes, heart, kidneys, cervix, and prostate. Internal illumination can also be used with optical contrast agents for functional and molecular imaging of internal tissues and organs inside the body. 103 With the development of relevant techniques, internal illumination is expected to play a critical role in clinical monitoring of cancer and diseases in internal organs. 7 PACT has matured greatly through biomedical research over the last two decades. Currently, PACT is capable of whole-body imaging of mice and organ imaging in mice, rats, pigs, and other animals. 4 PACT has also been frequently used in preclinical and clinical human body imaging, such as skin, breast, and extremity imaging. 115 Continuing efforts have been made to achieve PACT imaging of the human brain. We are confident that PACT will have a wide range of applications in biomedical research and clinical practice in the future.

CONFLICT OF INTEREST
The authors declare no potential conflicts of interest.

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DATA AVAILABILITY STATEMENT
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